Transplantation and immunosuppressive drugs. Transplantation is the treatment of choice for most patients with end stage kidney-failure, hearth or liver disease, autoimmune type 1 diabetes and it is a developing possibility for patients with deficiencies in small-bowel and lung function. Graft survival depends on a number of factors but the most significant of these is the administration of powerful immunosuppressive drugs. Transplantation between genetically disparate individuals evokes a rapid and potentially destructive alloreactive immune response that, if left uncontrolled, can lead to complete destruction of the transplanted organ. Administration of immunosuppressive drugs attenuates this response and thus prevents acute graft rejection. However, continued graft survival depends on life-long immunosuppression because withdrawal of immunosuppression results in re-activation of the rejection response, leading to rapid graft destruction.
Recently, among the immunosuppressive drugs, selective T cell inhibitors have been developed including cyclosporine A (CsA), FK506 and rapamycin. Both CsA and FK506 inhibit T cell activation by blocking calcineurin function and thereby prevent the generation of the potent nuclear factor of activated T cells (NFAT). This step is essential for up-regulating the mRNA of several cytokines, including IL-2. The major limitations of CsA and FK506 are their various toxicities. Moreover, both CsA and FK506 prevent T cell apoptosis (reviewed in Yu et al. 2001).
On the contrary, rapamycin is a potent immunosuppressant that inhibits T cell proliferation by binding a cytosolic protein (FKBP-12) and blocking IL-2 signaling (Sehgal 1998). The complex binds to and blocks the mammalian target of rapamycin (mTOR), resulting in the inhibition of cytokines induced T-cell proliferation. Importantly, in contrast to CsA and FK506, rapamycin does not block TCR-mediated T cell activation (Blaha et al, 2003) and IL-2 T cell priming for activation-induced cell death (AICD). This latter is a form of T cell apoptosis which seems to play a role in the induction of peripheral transplantation tolerance (Wells et al. 1999). Unlike CsA, which has no effects on dendritic cells (DC), rapamycin profoundly affects DC phenotype and function (Hackstein et al. 2002). It markedly reduces their antigen uptake capacity, thereby favoring the differentiation of DC with a tolerogenic phenotype. This effect, present at a low, physiologically relevant concentration of rapamycin (1 ng/ml) is independent of DC maturation and has been demonstrated both in vitro and in vivo (Hackstein et al. 2002).
Although the currently available immunosuppressive drugs are very effective in short term, substantial problems indicate a pressing need to develop alternative and more sophisticated ways of preventing graft rejection. The main obstacle is the inability to distinguish between beneficial immune responses against infectious pathogens and destructive immune responses against the graft. Thus, immunosuppressive therapies can lead to increased risk of opportunistic infections. Several studies show that non specific immunosuppression would lead to an increased incidence of cancer in transplanted patients (Hojo et al. 1999). Therefore, the full potential of transplantation will be fulfilled only when alternatives to non specific immunosuppression are found. The major aim of transplantation immunology is to develop protocols that prevent immune responses towards the graft but leave the rest of the immune system intact. This accomplishment will lead to transplantation tolerance.
Autoimmunity. In autoimmune diseases, undesired immune responses to self-antigens lead to destruction of peripheral tissues. Treatments of autoimmune diseases are currently based on downmodulation of inflammation and non-antigen (Ag) specific immunosuppression. As for prevention of allograft rejection, this strategy is frequently not effective in the long term with high risk of relapse once the drug is withdrawn and hazards of excessive immunosuppression, including infections and tumors. The alternative approach is based on the induction of transient immunosuppression and/or specific immune tolerance, aimed at “silencing” the pathogenic response to self-Ag, while keeping host defense mechanism intact.
The immune system has evolved two distinct mechanisms to induce tolerance to self or non-harmful antigens. These are referred to as central and peripheral T cell tolerance. Central tolerance is realised during fetal development and the very early natal period and is mediated by clonal deletion of self-reactive T cells during thymic development. Peripheral mechanisms induce tolerance in mature T cells and occur in the periphery during the whole life. These mechanisms include functional inactivation of antigen specific lymphocytes (named anergy) and activation of T cell subsets with suppressive and regulatory capacities (T regulatory cells reviewed in Battaglia et al. 2002),
Tolerance and T regulatory cells. Recently, there has been a growing interest in the induction of T regulatory (Tr) cells as a strategy to achieve graft specific tolerance. The majority of Tr cells identified to date lie within the CD4+ population, although other T cell subsets, such as CD8+, CD8+CD28− and TCR+CD4−CD8− have also been shown to contain cells with regulatory capacity. Within the CD4+ population, various fractions with suppressive properties have been identified. Our group has characterised a subset of Tr cells, defined as type 1 regulatory T cells (Tr1), which have a cytokine production profile distinct from that of Th1 and Th2 cells. Human and mouse Tr1 cells produce high levels of IL-10, significant amounts of IL-5, TGF-β, and IFN-γ, but low levels of IL-2 and no IL-4 (Groux et al. 1997). IL-10 is a crucial cytokine for the differentiation and effector functions of Tr1 cells. Culture of CD4+ T cells in the presence of antigen and IL-10 leads to generation of Tr1 cells that are able to suppress antigen-specific T cell responses in vitro and the development of autoimmune colitis in vivo (Groux et al. 1997). Tr1 cells can also be generated in vivo. Tr1 cells have indeed been isolated from peripheral blood of SCID-reconstituted patients, in whom high levels of IL-10 were associated with successful allogeneic stem cell transplantation (Bacchetta et al. 1994).
Tolerance and IL-10. IL-10 plays a key role in immunoregulation (reviewed in Moore et al. 2001). It inhibits proliferation and IL-2 production of T lymphocytes. IL-10 has strong anti-inflammatory properties by inhibiting production of pro-inflammatory cytokines such as TNF-α, IL-1, IL-6 and chemokines such as IL-8, MIP1α, and MIP1β by activated monocytes/macrophages, neutrophilis, eosinophilis, and mast cells. Moreover, IL-10 suppresses antigen-presenting capacities of antigen presenting cells such as monocytes/macrophages/DC by downregulating MHCII and co-stimulatory molecules. The ability of IL-10 to inhibit induction and effector function of T cell-mediated and anti-inflammatory immune responses led to numerous studies on IL-10 expression, function, and potential utility in bone marrow and organ transplantation. In studies of vascularized heart allograft in mice, IL-10 treatment of recipient animals prior to grafting enhanced graft survival, whereas providing IL-10 at or after the time of grafting had little beneficial effect or even enhanced rejection (Li et al. 1999). Patients exhibiting elevated levels of IL-10 production prior to BMT have lower incidence of GVHD and improved survival (Baker et al. 1999). On the contrary, high IL-10 levels in post-BMT GVHD patients indicates a poor prognosis for survival (Hempel 1997). However, Blazar and colleagues showed that treatment of mice with small amounts of IL-10 (10−3, 10−4 of the amount that increased mortality) protects against GVHD-associated lethality (Blazar et al. 1998).
Combination of immunosuppressive drugs with IL-10. The majority of immunosuppressive drugs in current clinical uses act by inhibiting T cell activation and thus prevents graft rejection. However, this may be counter-productive, as under appropriate circumstances, T cell activation may lead to the induction of processes facilitating the development of graft-specific tolerance. Therefore, the usage of immunosuppressive drugs might not be optimal when the aim is tolerance induction. A clear demonstration of this phenomenon comes from SCID patients in whom tolerance was achieved after allogeneic hematopoietic stem cell transplantation without any immunosuppressive therapy (Bacchetta et al. 1994). In these patients the presence of donor derived Tr1 cells specific for the host alloantigens correlated with stable mixed chimerism, high levels of IL-10 production in vivo, and normal immune functions in the absence of any immunosuppressive therapy. In contrast, in BMT patients who received an immunosuppressive regimen to control acute-GVHD, Tr1 cells could not be isolated from peripheral blood, although donor derived T cells specific for host alloantigens were detectable (Bacchetta et al. 1995).
Rapamycin represents a novel compound with interesting immuomodulatory properties. For this reason we combined the in vivo administration of rapamycin with IL-10 in order to prevent allograft rejection or modulate type 1 diabetes and to allow the in vivo development of Tr cells.